brain research 1631 (2016) 1–12

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report Psychotropic effects of plantarum PS128 in early life-stressed and naïve adult mice

Yen-Wenn Liua, Wei-Hsien Liua, Chien-Chen Wua,b, Yi-Chen Juana, n nn Yu-Chen Wua, Huei-Ping Tsaia, Sabrina Wangc, , Ying-Chieh Tsaia,b, aInstitute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan bProbiotic Research Center, National Yang-Ming University, Taipei 11221, Taiwan cInstitute of Anatomy and Cell Biology, National Yang-Ming University, 155, Section 2, Linong Street, Taipei 11221, Taiwan article info abstract

Article history: Ingestion of specific , namely “psychobiotics”, produces psychotropic effects on Accepted 12 November 2015 behavior and affects the hypothalamic–pituitary–adrenal axis and neurochemicals in the Available online 24 November 2015 brain. We examined the psychotropic effects of a potential psychobiotic bacterium, strain PS128 (PS128), on mice subjected to early life stress (ELS) Keywords: and on naïve adult mice. Behavioral tests revealed that chronic ingestion of PS128 Dopamine increased the locomotor activities in both ELS and naïve adult mice in the open field test. Early life stress In the elevated plus maze, PS128 significantly reduced the anxiety-like behaviors in naïve Hypothalamic–pituitary–adrenal adult mice but not in the ELS mice; whereas the depression-like behaviors were reduced in axis ELS mice but not in naïve mice in forced swimming test and sucrose preference test. PS128 PS128 administration also reduced ELS-induced elevation of serum corticosterone under both Psychobiotics basal and stressed states but had no effect on naïve mice. In addition, PS128 reduced Serotonin inflammatory cytokine levels and increased anti-inflammatory cytokine level in the serum of ELS mice. Furthermore, the dopamine level in the prefrontal cortex (PFC) was significantly increased in PS128 treated ELS and naïve adult mice whereas serotonin (5- HT) level was increased only in the naïve adult mice. These results suggest that chronic ingestion of PS128 could ameliorate anxiety- and depression-like behaviors and modulate neurochemicals related to affective disorders. Thus PS128 shows psychotropic properties and has great potential for improving stress-related symptoms. & 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Abbreviations: CFU, colony-forming unit; CORT, corticosterone; ELS, early life stress; EPM, elevated plus maze; FST, forced swimming test; HPA axis, hypothalamus–pituitary–adrenal axis; HPLC-ECD, high-performance liquid chromatography- electrochemical detection; IL, interleukin; LAB, lactic acid bacteria; MRS, de Man Rogosa and Sharpe; MS, maternal separation; NELS, non-ELS; OFT, open filed test; PD, postnatal day; SPT, sucrose preference test; TNF, tumor necrosis factor n Corresponding author. nn Corresponding author at: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, 155, Section 2, Linong Street, Taipei 11221, Taiwan. E-mail addresses: [email protected] (S. Wang), [email protected] (Y.-C. Tsai). http://dx.doi.org/10.1016/j.brainres.2015.11.018 0006-8993/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2 brain research 1631 (2016) 1–12

anxiety disorders (Green et al., 2010; McCrory et al., 2010; 1. Introduction Tyrka et al., 2013). Moreover, in animal studies MS has been shown to affect the development of serotonergic and dopa- fi Probiotics are living known to have bene - minergic systems and the HPA axis in the brain (Bravo et al., cial effects on the host when ingested in adequate amounts 2014; Rentesi et al., 2010). Thus, an animal model of MS is (FAO/WHO, 2001). Numerous studies have demonstrated the ideal for investigating the psychopathology of stress-related fi diverse bene ts of probiotics (Vyas and Ranganathan, 2012), disorders and for evaluating the psychotropic potentials of fl including anti-in ammatory effects (Liu et al., 2011), gut probiotics (O'Mahony et al., 2009). protection (Zareie et al., 2006), and the attenuation of meta- Although several probiotics showing psychotropic effects bolic dysfunctions (Barrett et al., 2012; Huang et al., 2013). In in animal models have been classified as psychobiotics, fl recent years, the in uences of probiotics on the central which are defined as probiotics that produce health benefits – nervous system (CNS) and behaviors via the microbiome in patients suffering from psychiatric illnesses, the field is – gut brain-axis have been uncovered (Collins et al., 2012). In still in its infancy (Dinan et al., 2013). In an attempt to screen particular, there is an association between the for potential psychobiotics in our lactic acid bacteria (LAB) and stress response, and the effects of probiotics on CNS bank, we administered several LAB strains by oral gavage to could be mediated by humoral, immune, neural, and meta- mice that underwent ELS and evaluated their psychotropic bolic pathways (Moloney et al., 2014; Sudo et al., 2004). Based potentials according to their ability to reverse depression-like on the roles of probiotics in regulating stress responses and behaviors. Among the strains tested, we found that Lactoba- maintaining CNS homeostasis, we think there is great poten- cillus plantarum strain PS128 (PS128), isolated from sponta- fi tial for utilizing bene cial bacteria to improve CNS-related neously fermented mustard greens in Taiwan, shows the abnormalities. highest potential. To our knowledge, the ability of this strain Several probiotics have been shown to have effects on to normalize stress-induced depression-like behaviors has normalizing stress-induced abnormal behaviors and regulat- not been reported previously. Therefore, in the current study – – ing the hypothalamus pituitary adrenal axis (HPA axis) and we further characterize its psychotropic effects on the HPA fl the in ammatory responses in animal models (Ait-Belgnaoui axis, immune responses, and neurochemical changes in the et al., 2012; Arseneault-Breard et al., 2012; Bercik et al., 2011; brain. In addition, we also administered PS128 to naïve adult Desbonnet et al., 2010). For example, pretreatment with mice to evaluate its effects on unstressed normal mice. probiotics in immune-deficient mice has been shown to normalize immune-mediated deficits in intestinal physiology and to affect CNS functions (Smith et al., 2014). Administra- 2. Results tion of Lactobacillus helveticus–containing probiotics reduces anxiety-like behaviors in rodents (Messaoudi et al., 2011; 2.1. PS128 causes distinct alterations in behavior in ELS Ohland et al., 2013). Both Bifidobacterium longum 1714 and and naïve adult mice Bifidobacterium breve 1205 reduce anxiety-like behavior in an anxious mouse strain (Savignac et al., 2014). It has also been We administered PS128 to ELS and naïve adult mice for reported that the administration of probiotics restores mono- 4 weeks and then started the behavioral tests to evaluate amine levels in key brain regions of rats in a maternal the behavioral effects of chronic PS128 ingestion. The beha- separation (MS) model of depression (Bercik et al., 2011; vioral tests were conducted starting from the least stressful Desbonnet et al., 2010). Also, probiotics have been shown to to the most stressful test in the following order: SPT, OFT, alter behavior and CNS function in naïve adult animals EPM, and FST. In the SPT, the ELS mice showed a clear (Moloney et al., 2014). These findings not only reveal the reduction in sucrose preference compared to NELS litter- complexity of gut–brain interactions upon stress challenge mates; chronic ingestion of PS128 could revert this reduction but also suggest that probiotics could be used to ameliorate (Fig. 1A). However, chronic ingestion of PS128 had no effect on stress-induced disorders. sucrose preference in the naïve mice (Fig. 1B). In the OFT Early life stress (ELS) is known to convey negative effects there was no difference in the total distance traveled by the on brain development and cause behavioral changes in ELS and NELS mice. However, the ELS mice showed a reduc- adulthood (Lupien et al., 2009). In rodents, MS is the most tion in the time spent in the center (Fig. 2A and B). Chronic commonly used method to create ELS for the study of the PS128 treatment in ELS mice significantly increased the total physiological and psychological impacts of ELS (O'Mahony distance traveled by the ELS mice but did not reverse the time et al., 2011; Sanchez et al., 2001). The affected animals show spent in the center by the mice (Fig. 2A and B); however, this long-lasting behavioral and physiological phenotypes, includ- treatment significantly increased the total distance traveled ing enhanced stress responses, anxiety-like and depression- and the time spent in the center by the naïve mice (Fig. 2C like behaviors, HPA axis hyperactivity, and abnormal neuro- and D). chemical changes (Cryan and Holmes, 2005). In addition, MS From the EPM test we observed that ELS mice spent also increases the immune response, as indicated by significantly more time in the closed arms than the NELS enhanced IL-6 release following concanavalin A (ConA) sti- group, indicating more anxiety-like behaviors in the ELS mice mulation (Desbonnet et al., 2010). In humans, ELS is also (Fig. 3A). Although we did not observe any change in the associated with immune dysregulation (Fagundes et al., PS128-treated ELS mice, this treatment significantly reduced 2013). Furthermore, ELS is a well-known risk factor for the closed arm time and increased open arm time of the psychopathology, including major depressive disorder and naïve mice (Fig. 3A and B). In contrast, in the FST, PS128 brain research 1631 (2016) 1–12 3

Fig. 1 – PS128 administration reversed the reduced sucrose preference in the ELS mice but has no effect on naïve mice. (A) ELS mice show a clear reduction in sucrose preference compared to NELS mice, and chronic PS128 ingestion reverts this reduction to a level similar to that of NELS mice. (B) Chronic PS128 ingestion has no effect on sucrose preference in naïve mice. ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128. Data in the ELS mice experiments were analyzed by one-way ANOVA; data in the naïve mice experiments were analyzed by t-test. ***po0.001.

Fig. 2 – Open field test indicates that chronic PS128 ingestion increases locomotor activities in both ELS and naïve mice and reduces anxiety-like behaviors in naïve mice. (A) In the open field test the total travel distance is increased in ELS mice after PS128 administration. (B) The time spent in the center area is significantly reduced in ELS mice, and PS128 administration had no effect on the center time of ELS mice. (C) The total travel distance in naïve mice is also increased following PS128 administration. (D) Time spent in the center area is significantly increased in naïve mice after chronic PS128 administration. ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128. Data in the ELS mice experiments were analyzed by one-way ANOVA; data in the naïve mice experiments were analyzed by t- test. *po0.05; ***po0.001. treatment significantly reduced the immobile time in the ELS corticosterone levels to access the HPA axis reactivity. During mice, reverting the immobility to a level similar to that of the the basal unstressed condition, the corticosterone level of the NELS group, whereas such treatment had no effect on the ELS mice was significantly higher than that of the NELS mice naïve mice (Fig. 4A and B). Taken together, chronic PS128 (Fig. 5A). Chronic PS128 treatment significantly reduced the treatment increased locomotor activity in both ELS mice and baseline corticosterone level in the ELS mice to a degree naïve rats (Fig. 2). For anxiety-like behavior evaluated by the similar to that of NELS mice (Fig. 5A). Under stressed condi- center time of OFT and open arm time of the EPM, PS128 had tions, in this case 30 min after forced swimming, ELS mice a significant effect on naïve mice but not ELS mice (Figs. 2 and also showed significantly higher corticosterone levels than 3). Conversely, PS128 significantly reduced depression-like did the NELS mice (Fig. 5A). Again, chronic PS128 treatment behaviors in ELS mice but had no effect on naïve mice could significantly reduce the elevated corticosterone level in (Figs. 1 and 4). stressed ELS mice (Fig. 5A). In the naïve mice, chronic 2.2. PS128 normalizes ELS-induced exaggerated ingestion of PS128 had no effect on the serum corticosterone corticosterone release level either at baseline or under stressed conditions (Fig. 5B). These results indicated that the HPA axis in the ELS mice was Maternal neglect is known to increase stress reactivity in dysregulated and administration of PS128 could normalize offspring (Meaney, 2001). Thus, we measured the serum the elevated corticosterone level (Fig. 5A). 4 brain research 1631 (2016) 1–12

Fig. 3 – Elevated plus maze test shows PS128 administration decreases anxiety-like behaviors in naïve mice but not ELS mice. (A) Time spent in closed arm is significantly increased in ELS mice and PS128 administration has no effect on ELS mice. (B) In naïve mice, PS128 administration significantly reduced the time spent in the closed arm and increased the time spent in the open arm. ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128. Data of closed or open arm in the ELS mice were analyzed separately by one-way ANOVA; data of closed or open arm in the naïve mice experiments were analyzed separately by t-test. *po0.05; ***po0.001.

Fig. 4 – Forced swim test indicates PS128 administration reduces depression-like behaviors in ELS mice but not in naïve mice. (A) The immobile time is significantly increased in ELS mice, and PS128 administration reduces the immobile time to the level similar to that of NELS mice. (B) PS128 administration has no effect on the immobile time of naïve mice. ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128. Data in the ELS mice experiments were analyzed by one-way ANOVA; data in the naïve mice experiments were analyzed by t-test. ***po0.001.

2.3. PS128 decreases ELS-induced inflammation the unstimulated culture there were no differences in TNF-α, IL-6, and IL-10 concentrations among different groups To evaluate the effects of PS128 on the immune system of ELS (Fig. 6D, E and F). When we stimulated the culture with ConA, mice, we measured the levels of the pro-inflammatory we found that splenocytes from ELS mice produced signifi- cytokines TNF-α and IL-6 and the anti-inflammatory cytokine cantly more TNF-α than splenocytes from NELS mice (Fig. 6D). IL-10 in serum and in mitogen-stimulated splenocytes. The In splenocytes from PS128-treated ELS mice, this enhance- ELS mice showed a significant reduction in serum TNF-α, ment was attenuated (Fig. 6D). The level of IL-6 in LPS- whereas the IL-6 level was increased compared with that of stimulated splenocytes from ELS mice was not different from NELS mice (Fig. 6A and B). The IL-10 concentration of ELS that of NELS mice; however, the IL-6 response of splenocytes mice seemed lower than that of the NELS mice; however, this from PS128-treated ELS mice was significantly higher than did not reach statistical significance (Fig. 6C). After chronic that of the other two groups (Fig. 6E). The levels of IL-10 in administration of PS128, the ELS mice showed no significant LPS-stimulated splenocytes from ELS mice showed a trend of change in TNF-α level (Fig. 6A). However, this treatment reduction; however, the difference was not statistically sig- significantly reduced the IL-6 level and increased the IL-10 nificant. The IL-10 levels of splenocytes from the PS128- level in the ELS mice (Fig. 6B and C). In the cultured treated ELS group were significantly higher than those of splenocytes, which were obtained from NELS, ELS, and the ELS mice (Fig. 6F). These results showed that PS128 PS128-treated ELS mice, we collected culture medium and modulated immune responses in ELS mice. However, PS128 measured the aforementioned cytokines. We found that in treatment showed no clear effects on cytokine levels in brain research 1631 (2016) 1–12 5

Fig. 5 – Chronic PS128 administration reduces corticosterone levels in ELS mice but not naïve mice. (A) ELS treatment significantly elevates blood corticosterone levels under both basal and stressed states. PS128 administration effectively reduces the corticosterone levels in the ELS mice to a level similar to that of NELS mice under both states. (B) PS128 administration has no effect on the corticosterone levels in naïve mice in both basal and stressed states. ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128. Data of basal or stressed state in the ELS mice experiments were analyzed separately by one-way ANOVA; data of basal or stressed state in the naïve mice experiments were analyzed separately by t-test. *po0.05; **po0.01; ***po0.001. serum or stimulated splenocytes from naïve mice (data not ratio of DOPAC to DA and HVA to DA were both significantly shown). lower than that of the untreated ELS mice (Table 1). In particular, the ratio of HVA to DA in PS128-treated mice 2.4. PS128 altered the serotonin and dopamine systems in was even lower than that of the NELS mice (Table 1). both ELS and naïve adult mice In naïve adult mice, PS128 treatment significantly increased the concentration of DA, DOPAC, and HVA Because ELS has been reported to increase serotonergic and (Table 2). However, the DA turnover rate was not different dopaminergic activities in the PFC, and to increase dopami- from that of the saline-treated mice (Table 2). In addition, nergic activity in the striatum (Rentesi et al., 2013), we also there was no change in the dopaminergic activity in the investigated whether PS128 could alter the ELS-induced striatum in ELS, PS128-treated ELS, and naïve mice groups changes in the serotonin and dopamine systems. We ana- (Tables 1 and 2). lyzed levels of 5-HT, DA, and their metabolites in the PFC and striatum by HPLC-ECD. In the PFC, ELS mice showed signifi- cantly reduced 5-HT levels and had no change in their 5-HIAA 3. Discussion concentration. As a result, the ratio of 5-HIAA to 5-HT was significantly increased in ELS mice compared to that of NELS In the present study we demonstrated the psychotropic mice, indicating an increase in serotonergic activity (Table 1). effects of PS128 on ELS and naïve adult mice. We used MS Chronic PS128 ingestion did not increase the reduced 5-HT in as the adverse event to create ELS. MS is a well-established ELS mice, but instead reduced the 5-HIAA concentration and paradigm used in rat models of ELS and has been shown to consequently reduced the ratio of 5-HIAA to 5-HT to the level result in prolonged and consistent dysfunctions in the gut– similar to that of NELS mice (Table 1). However, in the naïve brain-axis (O'Mahony et al., 2011). Hence, it proved to be a adult mice PS128 increased 5-HT concentration and useful platform for screening psychobiotics (Dinan et al., decreased both the 5-HIAA concentration and the ratio of 5- 2013). For that reason, we had adapted the rat MS procedure HIAA to 5-HT (Table 2). The serotonin system in the striatum with some modifications because our mice had ELS. It has was not significantly different in ELS, NELS, PS128-treated ELS been reviewed that MS in mice is difficult to work with mice, or naïve mice (Table 1). because it produces unreliable phenotypes that may be In the dopaminergic system, the DA concentration of PFC caused by either different stressor protocols or mice strains tended to decrease in ELS mice compared with NELS mice; (Millstein and Holmes, 2007; Savignac et al., 2011). However, however, this difference was not statistically significant we have consistently found that mice that underwent our (Table 1). The concentration of DA metabolite DOPAC was modified MS procedure developed reliable abnormal beha- not changed in ELS mice compared to NELS mice (Table 1). viors (Fig. 1). In addition to behavioral changes, we also found However, the ratio of DOPAC to DA was significantly higher dysfunctions in the HPA axis, immune response, and neuro- than that of the NELS mice (Table 1). Although the level of transmitters in the ELS mice. These findings suggest that the HVA was significantly reduced in ELS mice, the HVA-to-DA modified MS procedure could be used in mice for under- ratio was not significantly different from that of the NELS standing the adverse effects of ELS and for screening mice (Table 1). Following chronic PS128 ingestion, the treated psychobiotis. ELS mice had a significantly elevated DA level compared to Our ELS mice showed both anxiety-like and depression-like untreated ELS mice (Table 1). The DOPAC and HVA concen- phenotypes because they had reduced center time in OFT, trations of PS128-treated ELS mice were also significantly increased closed arm time in EPM, increased immobile time in higher than those of the untreated ELS mice. As a result, the the FST, and reduced preference for sucrose (Figs. 1–4). Most 6 brain research 1631 (2016) 1–12

Fig. 6 – Effects of PS128 administration on serum cytokines and cytokines released from stimulated splenocytes from ELS and naïve mice. (A) ELS treatment reduces serum TNF-α concentration, and PS128 administration has no effect on the TNF-α level of ELS mice. (B) Serum IL-6 level is significantly elevated in ELS mice, and chronic PS128 administration reversed this elevation. (C) Chronic PS128 administration increases serum levels of anti-inflammatory cytokine IL-10 in ELS mice. (D) ConA stimulation significantly induced TNF-α release in cultured splenocytes from ELS mice. Splenocytes from PS128-treated ELS mice show a significant TNF-α release. (E) IL-6 release is significantly elevated in splenocytes from PS128-treated ELS mice following LPS stimulation. (F) Splenocytes from ELS mice show reduced IL-10 release following LPS stimulation, whereas the IL-10 release in splenocytes from PS128-treated ELS mice is similar to that of NELS mice. ConA, concanavalin A; LPS, lipopolysaccharide; ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS. Data of serum cytokine levels in the ELS mice experiments were analyzed by one-way ANOVA; data of medium or ConA/LPS stimulated splenocyte experiments were analyzed separately by one-way ANOVA. *po0.05; **po0.01; ***po0.001. studies using MS show a single trait consisting of either suggest that our ELS procedure produces a more robust anxiety-like or depression-like behavior (as reviewed in behavioral phenotype with severely dysregulated stress Moloney et al., 2014). In addition, the serum corticosterone response. levels in our ELS mice were significantly higher in both basal In our ELS mice we found a significant increase in serum andstressedstates(Fig. 5). However, most of the MS animals in IL-6 levels accompanied by decreased serum IL-10 levels previous studies showed increased corticosterone levels only (Fig. 6). These changes have not been previously described during the stressed state (Rees et al., 2006). These results in MS rats (Desbonnet et al., 2010; O'Mahony et al., 2009). brain research 1631 (2016) 1–12 7

Table 1 – Alteration of 5-HT and DA neurochemicals in brain regions of ELS mice.

Prefrontal cortex Hippocampus Striatum

NELS ELS ELSþPS128 NELS ELS ELSþPS128 NELS ELS ELSþPS128

Monoamines and metabolites 5-HT 88.5745 42.3714*** 50.1730* 69.774.7 52.77 57.6724 5647109 6037156 6547120 5-HIAA 21.377.2 25.879.0 12.574.7**,### 62.5721 51.9718 39.7714 77.1743 98.7750 88.8757 DA 1687114 66.1733** 1907133## 36.0714 29.8724 33.0712 406971176 39317938 39997939 DOPAC 50.4711 59.9729 71.2728* 46.3721 36.279.0 34.8714 417795 4667224 333765 HVA 158751 129746 167771 72.7722 57.4710 59.0739 4597225 6877242 5197104

Turnover ratio 5-HIAA:5-HT 0.30170.38 0.61570.29** 0.22970.12### 0.93070.29 1.0870.55 0.73470.42 0.092470.020 0.18070.13 0.13070.058 DOPAC:DA 0.46870.28 0.83070.36** 0.41670.18### 1.0670.51 1.0470.36 0.93070.29 0.092370.0069 0.12370.064 0.085870.019 HVA:DA 1.6071.1 2.3171.2 0.97770.72## 1.8170.71 1.6870.54 1.4570.35 0.16170.016 0.17970.057 0.13370.028

Concentrations of monoamines, metabolites and turnovers (ng/g wet tissue) are expressed as mean7SEM. DA, dopamine; DOPAC, 3,4- dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HT, serotonin or 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid. Data were analyzed by one-way ANOVA. Statistically significant values are highlighted in gray. * po0.05; ** po0.01; *** po0.001 vs. NELS; ## po0.01; ### po0.001 vs. ELS.

Table 2 – Alterations of 5-HT and DA neurochemicals in brain regions of normal adult mice.

Prefrontal cortex Hippocampus Striatum

Saline PS128 Saline PS128 Saline PS128

Monoamines and metabolites 5-HT 47.8719 95.6735** 78.4718 99.6748 8297146 8687217 5-HIAA 25.3716 17.676.3 N/D N/D 65.3773 39.3750 DA 142766 3017157* 50.2720 62.8749 808071070 736471883 DOPAC 95.8739 121751 57.0712 65.0718 316748 351789 HVA 123737 147742 47.177.1 54.6720 521776 469793

Turnover ratio 5-HIAA:5-HT 0.63870.64 0.16070.037 N/D N/D 0.077070.040 0.078070.044 DOPAC:DA 0.69270.34 0.54070.22 1.3170.60 1.4770.67 0.039670.0077 0.045770.017 HVA:DA 0.82370.38 0.67970.28 1.0770.40 0.98170.53 0.064770.0084 0.066710.0051

Concentrations of monoamines, metabolites and turnovers (ng/g wet tissue) are expressed as mean7SEM. DA, dopamine; DOPAC, 3,4- dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryptamine or serotonin. Data were analyzed by t-test. Statistically significant values are highlighted in gray. N/D, not detected. * po0.05; ** po0.01 vs. Saline.

Elevation of serum IL-6 is associated with stress-related system (Moloney et al., 2014). Increased secretion of circulat- disorders in humans who had early adverse experiences ing glucocorticoids, including corticosterone, could inhibit during childhood (Coelho et al., 2014). It has been shown that both innate and adaptive immune responses (Sorrells and adults with childhood maltreatment and patients with Sapolsky, 2007). Thus, there may be a complex cross- depressive disorders have increased serum IL-6 levels regulation between the HPA axis and the immune system, (Carpenter et al., 2010; Pace et al., 2006). In addition, a clinical and this cross-regulation may lead to the modulation of study has demonstrated a higher ratio of serum IL-6 to IL-10 animal behavior. Sustained elevation of corticosterone might and a lower serum IL-10 level in patients with major depres- also result in the reduced serum TNF-α observed in the ELS sion compared to patients without major depression mice (Fig. 6A). (Dhabhar et al., 2009). Therefore, our data suggest that the In addition to these findings, concentrations of neuro- ELS in mice could induce an immune alternation that is transmitters DA and 5-HT were significantly reduced in the similar to changes found in clinical studies. Furthermore, the PFC of ELS mice, but there was an increase in the turnover HPA axis is also known to directly interact with the immune rate of both transmitters (Tables 1 and 2). The increased DA 8 brain research 1631 (2016) 1–12

and 5-HT turnover rate has also been reported in the The effects of PS128 on ELS mice are clearly different from maternal deprivation model in rats (Rentesi et al., 2013). that of the naïve mice. We found PS128 could reduce anxiety- The serotonergic system has long been linked to anxiety, like behaviors in naïve mice but not ELS mice (Fig. 3). How- and insufficient 5-HT concentration may lead to hyper- ever, the depression-like behaviors were reduced in PS128- excitability in the amygdala, a condition that is associated treated ELS mice but not naïve mice (Fig. 4). In addition, PS128 with adulthood anxiety (Etkin and Wager, 2007). Thus, treatment reduced serum corticosterone levels under both decreased 5-HT levels in the PFC may result in dysregulated basal and stressed conditions in ELS mice but had no effect excitability in the amygdala and consequently produce on naïve mice (Fig. 5). Furthermore, PS128 treatment anxiety-like behaviors in ELS mice (Fig. 3). Taken together, increased the 5-HT concentration in PFC only in naïve mice our ELS procedure produced mice with multiple deficits, (Table 2). Because the corticosterone response of the HPA axis including abnormal behaviors, HPA dysregulation, immune in naïve mice was not changed, the reduction of anxiety-like alternation, and changes in neurochemicals in the PFC. behaviors in naïve mice cannot be explained by regulating Following the daily gavage of PS128 for 4 weeks, we found the HPA axis response. Instead, it might be related to the that chronic administration of PS128 reduced the ELS- increased 5-HT level in the PFC for the reason mentioned induced depression-like behaviors, normalized the HPA axis previously, that is, that 5-HT could modulate the activity of and immune systems, and modulated the changes in the DA amygdala, which is associated with anxiety-like behavior and 5-HT system in the PFC. From previous studies, ELS is (Etkin and Wager, 2007). However, in ELS mice the reduced known to modulate gut microbiota and disrupt the integrity 5-HT concentration in PFC cannot be reversed by adminis- of the gut barrier, thus resulting in increased inflammation tration of PS128, and we did not see a reduction in anxiety- and abnormal behaviors (O'Mahony et al., 2011), both of like behavior (Fig. 3 and Table 1). which can be restored by the ingestion of beneficial bacteria. Administration of PS128 increased locomotor activity in Reports have shown that consumption of a commercially both ELS mice and naïve mice. Previous studies have shown available combination of that the administration of DA or its analog into specific brain R0011 and Lactobacillus helveticus R0052 increases the amount regions such as the striatum or the nucleus accumbens of gut lactobacilli, reduces permeability in the colon, and stimulates locomotion in animals (Mabrouk et al., 2014; reduces the elevated serum corticosterone induced by MS in Woodruff et al., 1976). Because we did not observe changes rat pups (Gareau et al., 2007). A recent study also shows that in the striatal dopaminergic activity (data not shown), this administration of the commensal bacterium Bacteroides fragi- locomotor enhancement probably did not involve the stria- lis improves gut barrier integrity and normalizes anxiety-like tum. However, a previous study has shown that the admin- behaviors in a mouse model known to display features of istration of Bifidobacterium infantis 35624 or citalopram to MS autism spectrum disorder (Hsiao et al., 2013). Also, adminis- rats modulated the level of noradrenaline in the brain stem tration of Lactobacillus farciminis has been shown to attenuate and normalized depression-like behavior (Desbonnet et al., gut hyperpermeability and the abnormal activation of the 2010). Changes in the noradrenaline system could also HPA axis induced by psychological stress (Ait-Belgnaoui et al., potentially modulate the locomotor activity and should be 2012). These results suggest that beneficial bacteria in the gut further explored in future studies of PS128. contribute to the improvement of the stress response. Thus, The most interesting finding in this study is that PS128 it is possible that PS128 normalizes the MS-induced dysregu- significantly reduced the hyperactive HPA axis response and lation of the HPA axis via modulating the gut microbiota and the depression-like behaviors in ELS mice (Figs. 1, 4 and 5). strengthening the gut barrier. We did not directly examine We also found an increased DA concentration and DA turn- the gut integrity of PS128-treated mice; however, judging by over rate in the PFC of PS128-treated ELS mice (Table 1). PFC is the lack of effect on levels of corticosterone in PS128-treated known to be a highly evolved brain region that is responsible naïve mice, which would have normal gut microbiota and gut for regulating working memory, decision-making, and emo- barrier functions, it is likely that the effect of PS128 on the tions, including stress response (Shansky and Lipps, 2013). HPA axis of ELS mice involves the improvement of gut Normal PFC function, especially regarding the reward/avoid- integrity. ance emotional responses, requires an adequate DA concen- Chronic treatment of PS128 also alleviated the increased tration (Arnsten, 2009). In addition, the mesocortical DA serum IL-6 concentration in ELS mice. The serum concentra- system may have a direct influence on the HPA axis tion of IL-6 is positively correlated with depression severity in (Feenstra et al., 1992). Thus, the hyperactive HPA axis and patients (Pace et al., 2006). Preclinical animal studies have depression-like behaviors seen in ELS mice might be directly shown that reduction of IL-6 is associated with reducing associated with their altered DA function in the PFC (Table 1). depression-like behaviors. For example, previous studies Interestingly, following PS128 administration, the DA system show that the administration of Bifidobacterium infantis 35624 of ELS mice was restored to the control level, and so were the to MS rats attenuated IL-6 release and reduced immobility in HPA axis responses and depression-like behaviors (Figs. 4, 5 FST (Desbonnet et al., 2010). Similarly, we found that chronic and Table 1). Hence, PS128 might suppress the exaggerated PS128 administration also reduced serum IL-6 and improved release of corticosterone and normalized depression-like depression-like behaviors in ELS mice (Figs. 4A and 6B). We behavior through increasing dopaminergic activity in the PFC. also found that administration of PS128 significantly In summary, we assessed the psychotropic effects of increases IL-6 production in LPS-stimulated splenocytes, PS128 in both ELS and naïve adult mice in an attempt to suggesting PS128 treatment could increase the function of identify novel psychobiotic strains. We demonstrate that the immune system against foreign pathogens (Fig. 6E). PS128 could normalize ELS-induced HPA axis hyperactivity brain research 1631 (2016) 1–12 9

and depression-like behaviors, and that in naïve mice it PD 29, only males pups were selected and were randomly reduced anxiety-like behaviors. Some of the ELS-induced assigned to the ELS experimental group (ELSþPS128, n¼10) serum cytokine and PFC neurochemical changes are also and ELS control group (ELS, n¼12). Mice without MS were restored by chronic PS128 administration. The psychotropic regarded as the no ELS control group (NELS; n¼10). effects of PS128 on ELS and naïve adult mice suggest great potential for PS128 to be used to improve affective behaviors 4.4. Experimental design and sample collection under both normal and diseased conditions. In addition, to our knowledge, PS128 is the first psychobiotic that increases To evaluate the psychotropic effects of PS128, we administered locomotor activity and modulates both serotonergic and PS128 to the ELS experimental group and to a separate naïve dopaminergic systems, which further expands its possible adult male group (naïve). The ELS and naïve experimental application in the treatment of psychiatric and neurological groups were given saline re-suspended PS128 daily (109 CFU/ disorders. mouse/day) by gavage for 4 weeks from PD 29 and from 8 weeks old, respectively, whereas the ELS and naïve control groups were given saline by gavage during the same period. At the end of the 4. Experimental procedure PS128 treatment period, when ELS group mice were 8 weeks old andnaïvegroupmicewere12weeksold,themiceunderwenta 4.1. Preparation of PS128 battery of behavioral tests. The tests were given in sequence from the least stressful to the most stressful in the following PS128 was isolated from fermented mustard greens, which is order: sucrose preference test (SPT); open field test (OFT); fi a traditional Hakka ethnic food product. It was identi ed as a elevated plus maze (EPM); and forced swimming test (FST). After fi novel strain by phylogenetic classi cation of its 16S rDNA the 4-day SPT the mice were subjected to OFT on the next day. sequence. The isolated PS128 was inoculated in Man Rogosa EPM test were conducted immediately after OFT on the same Sharpe broth (MRS; BD Difco, Becton-Dickinson, Sparks, MD test day. On the next day the first session of FST began. All the 1 MD, USA), cultured at 37 C for 18 h, and then harvested by tests were conducted during the light phase. centrifugation at 6000 g for 10 min. The pellet was re- On the last day of FST, before the swim start, the mice were fi suspended in MRS plus 12.5% glycerol to a nal concentration subjected to orbital sinus blood collection to obtain the basal 9 of 5 10 colony-forming units per milliliter. The re- state samples. Then, the mice underwent a 6-min forced swim suspended solution was then aliquoted in freezer tubes and and were returned to their home cage. After 30 min, blood 1 stored at 20 C until use. When in use the aliquot was pre- samples were collected again to obtain the stressed state 1 warmed to 37 C for 1 h and re-suspended in saline before samples. All blood sampling was conducted within the same being administered to mice. 3-hperiodinanefforttominimize the effect of circadian rhythm on corticosterone release. The collected whole blood was cen- 4.2. Animals and housing trifuged at 3000 g for 10 min at 4 1C, and then the serum was stored at 80 1C until use. Following blood collection, the mice Timed-pregnant female and naïve adult male C57BL/6J mice were sacrificed by cervical dislocation. The brains were quickly were purchased from the National Laboratory Animal Center removed and placed on dry ice. The ice-cold brain was dissected (Taipei, Taiwan). On arrival, the mice were accommodated in on a filter paper placed on a glass dish on ice. The prefrontal the specific pathogen-free room at the Laboratory Animal cortex (PFC), hippocampus, and striatum samples were dissected Center of National Yang-Ming University. The room was kept out and immediately preserved in ice-cold 0.6% perchloric acid at 2272 1C, 50–60% humidity, and under 12 h light/dark cycle. and stored at 80 1C until use. Spleen tissues were also The mice were provided with water and chow ad libitum collected, temporarily stored in ice-cold RPMI 1640 medium (LabDiet Autoclavable Rodent Diet 5010; PMI Inter- (Invitrogen, Carlsbad, CA, USA), and then processed in spleno- national, Brentwood, MO, USA). After 1 week of acclimation cyte experiments (see mitogen-stimulated splenocyte section). the experiments were started. All animal experimental pro- cedures were reviewed and approved by the Institutional 4.5. Sucrose preference test Animal Care and Use Committee, National Yang-Ming Uni- versity (IACUC No. 1001102). SPT was modified from a previous study by Yu et al. (2012).To habituate the mice to drinking sucrose-containing solution, 4.3. Maternal separation stress on the first day of the test the mice were given two bottles of 1% sucrose solution (g/mL) at 18:00 for 24 h. On the second We adapted MS procedures from a rat model of ELS described day, one of the sucrose bottles was replaced with tap water previously and made some modifications (Desbonnet et al., and left for 24 h. On the third day, the mice were water- 2010). Briefly, between postnatal day (PD) 2 and PD 14, male deprived for 18 h from 18:00 until 12:00 next day. On the and female neonates in the stress group were separated from fourth day, immediately after water deprivation, the mice their mothers and littermates and placed in a small glass were given one bottle of 1% sucrose and one bottle of tap bottle (5 cm in diameter) for 3 h per day (11:00–14:00) at room water for 5 h. The position of the bottles was switched to temperature without a heating pad (Supplementary Fig 1). avoid location preference. The sucrose and water consumed The no MS group was left with their mother undisturbed. For during the 5-h test were measured. The sucrose preference the next 2 weeks, all the mice were left undisturbed except (%) was calculated as follows: (weight of sucrose consumed/ for the routine bedding change and were weaned at PD 28. On total weight of sucrose and water consumed) 100. 10 brain research 1631 (2016) 1–12

4.6. Open field test ELISA kits (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Locomotor activity of the mice was examined by the OFT. In this test the mouse was placed in the open field activity 4.11. Mitogen-stimulated splenocytes chamber for 10 min (Tru Scan Activity System; Coulbourn Instruments, Whitehall, PA, USA). The square activity cham- Spleen preserved in ice-cold RPMI 1640 medium was first 3 ber (25.4 25.4 38 cm ) is made of Plexiglas walls with two squashed by a sterile piston and then filtered through a sieve photobeam sensor bars on each side. The box was cleaned mesh cell strainer (Becton, Dickinson and Company, East with 70% ethanol after each test. The activities were auto- Rutherford, NJ, USA) to isolate single cells. The preparation matically recorded and quantified with the Tru Scan 2.2 soft- was centrifuged at 1000 rpm for 5 min. The pellet was ware (Coulbourn Instruments). The total distance traveled, collected and treated with lysis buffer for 5 min to remove moving time, and center distance and time were measured by erythrocyte. Then, the preparation was centrifuged again to the Tru Scan Activity System. The center area was defined as remove lysis buffer and washed in serum-free RPMI 1640 2 a region in the center measuring 12.5 12.5 cm . medium. After the final centrifugation to remove serum-free medium, the splenocyte pellet was re-suspended in RPMI 4.7. Elevated plus maze test 1640 medium plus 10% fetal bovine serum and seeded at 2 106 cells/200 μL/well in 96-well flat-bottom plates. The The elevated plus maze is composed of two closed arms and two stimulated splenocytes were cultured with mitogen conca- open arms (height, 45 cm; full arm length, 66 cm; arm width, navalin A (ConA; 4 μg/mL) or lipopolysaccharide (LPS; 10 cm; wall height of closed arm, 30 cm). This test was used to μ 1 0.6 g/mL) in the culture medium for 48 h at 37 C (5% CO2). assess anxiety-like behavior of the mice. The mouse was placed The control splenocytes were cultured for the same period of 2 in the center arm crossing area (10 10 cm )andallowedfree time without mitogens. exploration in the maze for 10 min. The maze was cleaned with 70% ethanol after each test. The mouse activity was recorded by 4.12. Quantification of monoamines and metabolites a video camera mounted on the ceiling of the maze center. The recorded activity was later analyzed by EthoVision video tracking The frozen PFCs, hippocampus, and striata were thawed and software (Noldus Information Technology, Wageningen, the homogenized with a micro-sonicator (Q125 sonicator; Qsonica, Netherlands). The total travel distance and duration spent in Newton, CT, USA). The homogenized samples were then cen- the open and closed arms were quantified. trifuged at 12,000 g for 10 min at 4 1C. The supernatants were filtered through a 0.22-mm polyvinylidene difluoride membrane 4.8. Forced swimming test (4-mm syringe filter; Millex-GV; Millipore, Billerica, MA, USA). Afterproperdilutionwetook20-μl samples to measure the The FST was used to assess the depression-like behaviors in the concentrations of monoamines and their metabolites by the mice. Briefly, mice were put in a transparent acrylic cylinder high-performance liquid chromatography-electrochemical (height, 30 cm; internal diameter, 10 cm) containing 15 cm water detection system (HPLC-ECD). The HPLC-ECD comprised a (23–25 1C) to swim for 6 min on the first day. After the swim, the micropump (CMA-100; CMA, Stockholm, Sweden), an online mice were dried with tissue paper and returned to their home injector (CMA-160), a Microtech LC-pump (Microtech Scientific, cage. The next day, the mice were given a second swim session Sunnyvale, CA, USA), a BAS-4C electrochemical detector (Bioa- for 5 min. Only naïve adult mice were subjected to the first and nalytical Systems, Inc., West Lafeyette, IN, USA), and a reversed- second sessions of FST. Mice in the ELS experiments underwent phase column (Kinetex C ,2.6μm, 100 2.1 mm I.D.; Phenom- only the first day session. The swim sessions were recorded by a 18 enex, Torrance, CA, USA) as previously described (Cheng et al., video camera and the behavior was analyzed by EthoVision video tracking software. The immobile time were quantified 2000). The potential for the glassy carbon working electrode was þ from the first day session of ELS groups and from the second day set at 650 mV with respect to an Ag/AgCl reference electrode at 1 session of naïve adult mice. room temperature (25 C). The mobile phase containing 0.1 M NaH2PO4, 8% methanol, 0.74 mM SOS (1-octanesulfonic acid, 4.9. Serum corticosterone salt), 0.03 mM EDTA, and 2 mM KCl was adjusted to pH fi μ 3.74 with H3PO4.Diluted ltrates were then injected (20 L) into fl The serum was diluted first, and then we used a commercial the chromatographic system at a ow rate of 0.2 mL/min. CORT EIA kit (Cayman Chemical, Michigan, MI, USA) to Concentrations of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), analyze corticosterone concentration. The corticosterone homovanillic acid (HVA), 5-HT, and 5-hydroxyindoleacetic acid concentration was interpolated using the standards provided (5-HIAA) in the samples were interpolated using standards in the kit following the manufacturer’s instructions. (Sigma-Aldrich, St. Louis, MO, USA) ranging from 1 to 100 ng/mL.

4.10. Serum cytokine 4.13. Statistical analysis

We measured cytokine levels in the serum samples collected All data were expressed as mean7SEM. Differences between during the baseline period and in the culture medium of groups were analyzed by one-way or two-way analysis of stimulated splenocytes. Tumor necrosis factor (TNF)-α, inter- variance (ANOVA) with Bonferroni’s post-test or two-tailed t- leukin (IL)-6, and IL-10 were measured using commercial test when appropriate. brain research 1631 (2016) 1–12 11

gut–brain communication. Neurogastroenterol. Motil. 23, Contributions 1132–1139. Bravo, J.A., Dinan, T.G., Cryan, J.F., 2014. Early-life stress induces WSL, YCJ, and YCT designed the study. WSL, YCJ, YCW, and persistent alterations in 5-HT1A receptor and serotonin HPT performed the experiments. WSL, SW, and YCJ analyzed transporter mRNA expression in the adult rat brain. Front. Mol. Neurosci. 7, 24. and interpreted the data. YWL, SW, WSL, CCW, YCJ, and YCT Carpenter, L.L., Gawuga, C.E., Tyrka, A.R., Lee, J.K., Anderson, G. wrote the manuscript. M., Price, L.H., 2010. Association between plasma IL-6 response to acute stress and early-life adversity in healthy adults. Neuropsychopharmacology 35, 2617–2623. Disclosure Cheng, F.C., Kuo, J.S., Huang, H.M., Yang, D.Y., Wu, T.F., Tsai, T.H., 2000. Determination of catecholamines in pheochromocy- The authors have declared no conflict of interest related to toma cell (PC-12) culture medium by microdialysis-microbore this study. liquid chromatography. J. Chromatogr. A 870, 405–411. Coelho, R., Viola, T.W., Walss-Bass, C., Brietzke, E., Grassi-Oliveira, R., 2014. Childhood maltreatment and inflammatory markers: Acknowledgments a systematic review. Acta Psychiatr. Scand. 129, 180–192. Collins, S.M., Surette, M., Bercik, P., 2012. The interplay between the intestinal microbiota and the brain. Nat. Rev. Microbiol. 10, We thank Prof. Tung-Hu Tsai for providing the HPLC-ECD 735–742. system, Ko-Fan Lu and Heng-Chun Lin for contributing part Cryan, J.F., Holmes, A., 2005. The ascent of mouse: advances in of the work, and Dr. Wang-Tso Lee and Dr. Cheng-Jee Hong modelling human depression and anxiety. Nat. Rev. Drug for helpful discussions. This work was supported by the Discov. 4, 775–790. Academic Technology Development Program (101-EC-17-A- Desbonnet, L., Garrett, L., Clarke, G., Kiely, B., Cryan, J.F., Dinan, T. fi 17-S1-197) of the Ministry of Economic Affairs, Republic of G., 2010. Effects of the probiotic Bi dobacterium infantis in the maternal separation model of depression. Neuroscience 170, China. The funding source had no contribution to the study 1179–1188. design, collection, analysis, interpretation of the data, or Dhabhar, F.S., Burke, H.M., Epel, E.S., Mellon, S.H., Rosser, R., Reus, writing of the report for publication. V.I., Wolkowitz, O.M., 2009. Low serum IL-10 concentrations and loss of regulatory association between IL-6 and IL-10 in adults with major depression. J. Psychiatr. Res. 43, 962–969. Appendix A. Supplementary material Dinan, T.G., Stanton, C., Cryan, J.F., 2013. Psychobiotics: a novel class of psychotropic. Biol. Psychiatry 74, 720–726. Supplementary data associated with this article can be found Etkin, A., Wager, T.D., 2007. Functional neuroimaging of anxiety: a in the online version at http://dx.doi.org/10.1016/j.brainres. meta-analysis of emotional processing in PTSD, social anxiety disorder, and specific phobia. Am. J. Psychiatry 164, 1476–1488. 2015.11.018. Fagundes, C.P., Glaser, R., Kiecolt-Glaser, J.K., 2013. Stressful early life experiences and immune dysregulation across the life- span. Brain Behav. Immun. 27, 8–12. references FAO/WHO, 2001. Health and Nutritional Properties of Probiotics in Food Including Powder Milk With Live Lactic Acid Bacteria. Cordoba, Argentina. Ait-Belgnaoui, A., Durand, H., Cartier, C., Chaumaz, G., Eutamene, Feenstra, M.G., Kalsbeek, A., van Galen, H., 1992. Neonatal lesions H., Ferrier, L., Houdeau, E., Fioramonti, J., Bueno, L., Theo- of the ventral tegmental area affect monoaminergic responses dorou, V., 2012. Prevention of gut leakiness by a probiotic to stress in the medial prefrontal cortex and other dopamine treatment leads to attenuated HPA response to an acute projection areas in adulthood. Brain Res. 596, 169–182. psychological stress in rats. Psychoneuroendocrinology 37, Gareau, M.G., Jury, J., MacQueen, G., Sherman, P.M., Perdue, M.H., 1885–1895. 2007. Probiotic treatment of rat pups normalises corticoster- Arnsten, A.F., 2009. Stress signalling pathways that impair pre- one release and ameliorates colonic dysfunction induced by frontal cortex structure and function. Nat. Rev. Neurosci. 10, maternal separation. Gut 56, 1522–1528. 410–422. Green, E.K., Grozeva, D., Jones, I., Jones, L., Kirov, G., Caesar, S., Arseneault-Breard, J., Rondeau, I., Gilbert, K., Girard, S.A., Tomp- Gordon-Smith, K., Fraser, C., Forty, L., Russell, E., Hamshere, kins, T.A., Godbout, R., Rousseau, G., 2012. Combination of M.L., Moskvina, V., Nikolov, I., Farmer, A., McGuffin, P., Well- Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 come Trust Case Control, C., Holmans, P.A., Owen, M.J., reduces post-myocardial infarction depression symptoms and O’Donovan, M.C., Craddock, N., 2010. The bipolar disorder risk restores intestinal permeability in a rat model. Br. J. Nutr. 107, allele at CACNA1C also confers risk of recurrent major 1793–1799. depression and of schizophrenia. Mol. Psychiatry 15, Barrett, E., Fitzgerald, P., Dinan, T.G., Cryan, J.F., Ross, R.P., Quigley, 1016–1022. E.M., Shanahan, F., Kiely, B., Fitzgerald, G.F., O’Toole, P.W., Hsiao, E.Y., McBride, S.W., Hsien, S., Sharon, G., Hyde, E.R., McCue, Stanton, C., 2012. Bifidobacterium breve with alpha-linolenic T., Codelli, J.A., Chow, J., Reisman, S.E., Petrosino, J.F., Patter- acid and linoleic acid alters fatty acid metabolism in the son, P.H., Mazmanian, S.K., 2013. Microbiota modulate beha- maternal separation model of irritable bowel syndrome. PLoS vioral and physiological abnormalities associated with One 7, e48159. neurodevelopmental disorders. Cell 155, 1451–1463. Bercik, P., Park, A.J., Sinclair, D., Khoshdel, A., Lu, J., Huang, X., Huang, H.Y., Korivi, M., Tsai, C.H., Yang, J.H., Tsai, Y.C., 2013. Deng, Y., Blennerhassett, P.A., Fahnestock, M., Moine, D., Supplementation of Lactobacillus plantarum K68 and fruit– Berger, B., Huizinga, J.D., Kunze, W., McLean, P.G., Bergonzelli, vegetable ferment along with high fat–fructose diet attenu- G.E., Collins, S.M., Verdu, E.F., 2011. The anxiolytic effect of ates metabolic syndrome in rats with insulin resistance. Evid. Bifidobacterium longum NCC3001 involves vagal pathways for Based Complement. Altern. Med. 2013, 943020. 12 brain research 1631 (2016) 1–12

Liu, Y.W., Su, Y.W., Ong, W.K., Cheng, T.H., Tsai, Y.C., 2011. Oral Rentesi, G., Antoniou, K., Marselos, M., Syrrou, M., Papadopoulou- administration of Lactobacillus plantarum K68 ameliorates DSS- Daifoti, Z., Konstandi, M., 2013. Early maternal deprivation- induced ulcerative colitis in BALB/c mice via the anti- induced modifications in the neurobiological, neurochemical inflammatory and immunomodulatory activities. Int. Immu- and behavioral profile of adult rats. Behav. Brain Res. 244, nopharmacol. 11, 2159–2166. 29–37. Lupien, S.J., McEwen, B.S., Gunnar, M.R., Heim, C., 2009. Effects of Sanchez, M.M., Ladd, C.O., Plotsky, P.M., 2001. Early adverse stress throughout the lifespan on the brain, behaviour and experience as a developmental risk factor for later psycho- cognition. Nat. Rev. Neurosci. 10, 434–445. pathology: evidence from rodent and primate models. Dev. Mabrouk, O.S., Semaan, D.Z., Mikelman, S., Gnegy, M.E., Kennedy, Psychopathol. 13, 419–449. R.T., 2014. Amphetamine stimulates movement through tha- Savignac, H.M., Dinan, T.G., Cryan, J.F., 2011. Resistance to early- lamocortical glutamate release. J. Neurochem. 128, 152–161. life stress in mice: effects of genetic background and stress McCrory, E., De Brito, S.A., Viding, E., 2010. Research review: the duration. Front. Behav. Neurosci. 5, 13. neurobiology and genetics of maltreatment and adversity. J. Savignac, H.M., Kiely, B., Dinan, T.G., Cryan, J.F., 2014. Bifidobac- Child Psychol. Psychiatry 51, 1079–1095. teria exert strain-specific effects on stress-related behavior Meaney, M.J., 2001. Maternal care, gene expression, and the and physiology in BALB/c mice. Neurogastroenterol. Motil. 26, transmission of individual differences in stress reactivity 1615–1627. across generations. Annu. Rev. Neurosci. 24, 1161–1192. Shansky, R.M., Lipps, J., 2013. Stress-induced cognitive dysfunc- Messaoudi, M., Lalonde, R., Violle, N., Javelot, H., Desor, D., Nejdi, tion: hormone-neurotransmitter interactions in the prefrontal A., Bisson, J.F., Rougeot, C., Pichelin, M., Cazaubiel, M., cortex. Front. Hum. Neurosci. 7, 123. Cazaubiel, J.M., 2011. Assessment of psychotropic-like prop- Smith, C.J., Emge, J.R., Berzins, K., Lung, L., Khamishon, R., Shah, erties of a probiotic formulation (Lactobacillus helveticus R0052 P., Rodrigues, D.M., Sousa, A.J., Reardon, C., Sherman, P.M., and Bifidobacterium longum R0175) in rats and human subjects. Barrett, K.E., Gareau, M.G., 2014. Probiotics normalize the gut– Br. J. Nutr. 105, 755–764. brain–microbiota axis in immunodeficient mice. Am. J. Phy- Millstein, R.A., Holmes, A., 2007. Effects of repeated maternal siol. Gastrointest. Liver Physiol. 307, G793–G802. separation on anxiety- and depression-related phenotypes in Sorrells, S.F., Sapolsky, R.M., 2007. An inflammatory review of different mouse strains. Neurosci. Biobehav. Rev. 31, 3–17. glucocorticoid actions in the CNS. Brain Behav. Immun. 21, Moloney, R.D., Desbonnet, L., Clarke, G., Dinan, T.G., Cryan, J.F., 259–272. 2014. The microbiome: stress, health and disease. Mamm. Sudo, N., Chida, Y., Aiba, Y., Sonoda, J., Oyama, N., Yu, X.N., Kubo, Genome 25, 49–74. C., Koga, Y., 2004. Postnatal microbial colonization programs O’Mahony, S.M., Marchesi, J.R., Scully, P., Codling, C., Ceolho, A.M., the hypothalamic-pituitary-adrenal system for stress Quigley, E.M., Cryan, J.F., Dinan, T.G., 2009. Early life stress response in mice. J. Physiol. 558, 263–275. alters behavior, immunity, and microbiota in rats: implica- Tyrka, A.R., Burgers, D.E., Philip, N.S., Price, L.H., Carpenter, L.L., tions for irritable bowel syndrome and psychiatric illnesses. 2013. The neurobiological correlates of childhood adversity Biol. Psychiatry 65, 263–267. and implications for treatment. Acta Psychiatr. Scand. 128, O’Mahony, S.M., Hyland, N.P., Dinan, T.G., Cryan, J.F., 2011. 434–447. Maternal separation as a model of brain–gut axis dysfunction. Vyas, U., Ranganathan, N., 2012. Probiotics, prebiotics, and Psychopharmacology 214, 71–88. synbiotics: gut and beyond. Gastroenterol. Res. Pract. 2012, Ohland, C.L., Kish, L., Bell, H., Thiesen, A., Hotte, N., Pankiv, E., 872716. Madsen, K.L., 2013. Effects of Lactobacillus helveticus on murine Woodruff, G.N., Kelly, P.H., Elkhawad, A.O., 1976. Effects of behavior are dependent on diet and genotype and correlate dopamine receptor stimulants on locomotor activity of rats with alterations in the gut microbiome. Psychoneuroendocri- with electrolytic or 6-hydroxydopamine-induced lesions of nology 38, 1738–1747. the nucleus accumbens. Psychopharmacologia 47, 195–198. Pace, T.W., Mletzko, T.C., Alagbe, O., Musselman, D.L., Nemeroff, Yu, H., Wang, D.D., Wang, Y., Liu, T., Lee, F.S., Chen, Z.Y., 2012. C.B., Miller, A.H., Heim, C.M., 2006. Increased stress-induced Variant brain-derived neurotrophic factor Val66Met poly- inflammatory responses in male patients with major depres- morphism alters vulnerability to stress and response to sion and increased early life stress. Am. J. Psychiatry 163, antidepressants. J. Neurosci. 32, 4092–4101. 1630–1633. Zareie, M., Johnson-Henry, K., Jury, J., Yang, P.C., Ngan, B.Y., Rees, S.L., Steiner, M., Fleming, A.S., 2006. Early deprivation, but McKay, D.M., Soderholm, J.D., Perdue, M.H., Sherman, P.M., not maternal separation, attenuates rise in corticosterone 2006. Probiotics prevent bacterial translocation and improve levels after exposure to a novel environment in both juvenile intestinal barrier function in rats following chronic psycho- and adult female rats. Behav. Brain Res. 175, 383–391. logical stress. Gut 55, 1553–1560. Rentesi, G., Antoniou, K., Marselos, M., Fotopoulos, A., Alboy- charali, J., Konstandi, M., 2010. Long-term consequences of early maternal deprivation in serotonergic activity and HPA function in adult rat. Neurosci. Lett. 480, 7–11.